The Ig? locus produces a wide array of Ig kappa light chain rearrangements, but how this complex process is regulated is not known. The creation of a diverse Ig? repertoire is facilitated by major changes in the 3-dimensional structure of the Ig? locus called locus contraction that occur at the time of rearrangement. This compaction allows V? genes throughout the locus a chance to come into proximity with the J? genes to which one V? gene will rearrange in each pre-B cell. However, the factors which are important in orchestrating the 3D structure and long-range looping interactions of the Ig? locus in pre-B cells are not known. Furthermore, how the 3D structure affects V? gene utilization is not known. We have performed ChIP-seq in RAG-/-?+ pre-B cells for a variety of histone posttranslational modifications, including H3K4me1, the characteristic epigenetic mark of enhancers. It was surprising to observe that there were so many peaks of H3K4me1 within the V? locus, most of which are not present in pro-B cells. This developmental stage-specificity of the H3K4me1 regions suggests that these regions may play a role in regulating V? rearrangement. Published studies indicate that there are some regions within the V? locus that are preferential sites of long-range interactions with iE?, the enhancer located just downstream of the 4 J? genes. We found that the most predominant long-range interaction hubs are characterized by broad regions of high H3K4me1, and ChIP-seq reveals that these hubs demonstrate binding of multiple key transcription factors (TF) such as Pax5, EBF, E2A, Ikaros, PU.1 or YY1. We therefore hypothesize that some of these putative novel regulatory elements will be important in regulating the composition of the Ig? repertoire, possibly by orchestrating the 3D configuration of the contracted Ig? locus. We will assay these regions for traditional enhancer activity in a pGL3 luciferase vector. Specific TF binding sites within the enhancer regions will be mutagenized to determine which TF are essential for enhancer activity. To test our hypothesis that these regulatory regions will influence V gene repertoire composition, possibly by altering the 3D structure of the locus, we will delete putative regulatory regions, and also mutate individual TF binding sites within these regions, in an Abelson pre-B cell line, using CRISPR/Cas9 PrecisionX homologous recombination genome editing technology. Culture of the Abl pre-B cell line with the Abl-kinase inhibitor STI571 for 48 hours robustly induces a diverse repertoire of Ig? rearrangements. The effects of the deletions or mutations on long-range interactions will be assayed by 3C, and changes in the Ig? repertoire will be assayed by TaqMan real time PCR and by deep sequencing. We hypothesize that deletion of key TF binding sites and/or larger regulatory regions with binding sites for several key TF will profoundly affect the resulting Ig? repertoire. Whether this effect will be predominantly of genes in the vicinity of each enhancer/TF binding site cluster, or more globally, will be determined. This would demonstrate a highly novel function for these elements bearing the epigenetic characteristics of traditional enhancers.